Many plants, bacteria and marine algae accumulate quaternary ammonium compounds (QACs) in response to abiotic stresses such as drought and salinity. Gorham J (1991) Betaines in higher plants—biosynthesis and role in stress metabolism. In R M Wallsgrove, ed., Amino acids and their derivatives in higher plants. Cambridge University Press, Cambridge, pp 173–203. QACs can accumulate to high concentrations to increase the osmotic pressure of the cytoplasm without perturbing metabolism. Yancey P H (1994) Compatible and counteracting solutes. In K. Strange, ed., Cellular and molecular physiology of cell volume regulation. CRC Press, Boca Raton, Fla. pp 81–109 They also stabilize enzymes and membranes. Id. The synthetic pathway to glycine betaine, the most common QAC, has therefore been the target of recent metabolic engineering efforts to improve plant stress tolerance. McNeil et al., Plant Physiol 120:945–949, 1999; Rathinasabapathi, Ann Bot 86:709–716, 2000; Sakamoto and Murata, J Exp Bot 51:81–88, 2000. However, these efforts have met with only limited success due to metabolic constraints on the availability of the precursor choline. Hayashi et al., Plant J 12:133–142, 1997; Nuccio et al., Plant J 12:133–142, 1998; Huang et al., Plant Physiol 122:747–756, 2000.
Most members of the highly stress-tolerant plant family Plumbaginaceae accumulate β-alanine betaine instead of glycine betaine. Hanson et al., Plant Physiol 97:1199–1205, 1991; Hanson et al., Proc Natl Acad Sci USA 91:306–310, 1994. It was proposed that β-alanine betaine is a more suitable osmoprotectant than glycine betaine under saline hypoxic conditions since the first step in glycine betaine synthesis requires molecular oxygen. Id. Further, β-alanine betaine accumulation was proposed to be an evolutionary strategy to avoid metabolic limitations for choline (Hanson et al., Proc Natl Acad Sci USA 91:306–310, 1994) since β-alanine betaine is synthesized from the ubiquitous primary metabolite β-alanine.
To further investigate the synthesis and biological significance of β-alanine betaine, radiotracer experiments were conducted. These experiments showed that β-alanine betaine is synthesized by S-adenosyl-L-methionine (AdoMet) dependent N-methylation of β-alanine via N-methyl β-alanine and N,N-dimethyl β-alanine (Rathinasabapathi et al., Physiol Plant 109: 225–231, 2000; FIG. 1). Using a rapid and sensitive radiometric assay, AdoMet dependent N-methyltransferase (NMTase) activities were demonstrated in β-alanine betaine accumulating members of the Plumbaginaceae family (Rathinasabapathi et al., Physiol Plant 109: 225–231, 2000). Heretofore, however, the gene encoding the NMTase was not cloned, and the protein responsible for the NMTase activities was uncharacterized.